The present invention relates generally to plant molecular biology. More specifically, it relates to nucleic acids and methods for modulating their expression in plants.
Cell division plays a crucial role during all phases of plant development. The continuation of organogenesis and growth responses to a changing environment requires precise spatial, temporal and developmental regulation of cell division activity in meristems (and in cells with the capability to form new meristems such as in lateral root formation). Such control of cell division is also important in organs themselves (i.e. separate from meristems per se), for example, in leaf expansion, secondary growth, and endoreduplication.
A complex network controls cell proliferation in eukaryotes. Various regulatory pathways communicate environmental constraints, such as nutrient availability, mitogenic signals such as growth factors or hormones, or developmental cues such as the transition from vegetative to reproductive. Ultimately, these regulatory pathways control the timing, frequency (rate), plane and position of cell divisions.
Plants have unique developmental features that distinguish them from other eukaryotes. Plant cells do not migrate, and thus only cell division, expansion and programmed cell death affect morphogenesis. Organs are formed throughout the entire life span of the plant from specialized regions called meristems. In addition, many differentiated cells have the potential to both dedifferentiate and to reenter the cell cycle. There are also numerous examples of plant cell types that undergo endoreduplication, a process involving nuclear multiplication without cytokinesis. The study of plant cell cycle control genes is expected to contribute to the understanding of these unique phenomena. (O. Shaul et al., Regulation of Cell Division in Arabidopsis, Critical Reviews in Plant Sciences, 15(2):97-112 (1996)).
Current transformation technology provides an opportunity to engineer plants with desired traits. Major advances in plant transformation have occurred over the last few years. However, in many major crop plants, serious genotype limitations still exist. Transformation of some agronomically important crop plants continues to be both difficult and time consuming. For example, it is difficult to obtain a culture response from some maize varieties. Typically, a suitable culture response has been obtained by optimizing medium components and/or explant material and source. This has led to success in some genotypes. While, transformation of model genotypes is efficient, the process of introgressing transgenes into production inbreds is laborious, expensive and time consuming. It would save considerable time and money if genes could be introduced into and evaluated directly in commercial hybrids.
There is evidence to suggest that cells must be dividing for transformation to occur. It has also been observed that dividing cells represent only a fraction of cells that transiently express a transgene. Furthermore, the presence of damaged DNA in non-plant systems (similar to DNA introduced by particle gun or other physical means) has been well documented to rapidly induce cell cycle arrest (W. Siede, Cell cycle arrest in response to DNA damage: lessons from yeast, Mutation Res. 337(2:73-84).
Current methods for genetic engineering in maize require a specific cell type as the recipient of new DNA. These cells are found in relatively undifferentiated, rapidly growing callus cells or on the scutellar surface of the immature embryo (which gives rise to callus). Irrespective of the delivery method currently used, DNA is introduced into literally thousands of cells, yet transformants are recovered at frequencies of 10xe2x88x925 relative to transiently-expressing cells. Exacerbating this problem, the trauma that accompanies DNA introduction directs recipient cells into cell cycle arrest and accumulating evidence suggests that many of these cells are directed into apoptosis or programmed cell death. (Reference Bowen et al., International Plant Mol. Biol. Meetings, Tucson, Ariz. 1991). Therefore it would be desirable to provide improved methods capable of increasing transformation efficiency in a number of cell types.
In spite of increases in yield and harvested area worldwide, it is predicted that over the next ten years, meeting the demand for corn will require an additional 20% increase over current production (Dowswell, C. R., Paliwal, R. L., Cantrell, R. P., 1996, Maize in the Third World, Westview Press, Boulder, Colo.).
The components most often associated with maize productivity are grain yield or whole-plant harvest for animal feed (in the forms of silage, fodder, or stover). Thus the relative growth of the vegetative or reproductive organs might be preferred, depending on the ultimate use of the crop. Whether the whole plant or the ear are harvested, overall yield will depend strongly on vigor and growth rate. It would therefore be valuable to develop new methods that contribute to the increase in crop yield.
The proteins encoded by the wee1 polynucleotides range from approximately 107 kD in Saccharomyces cerevisiae to 68 kD in Xenopus. The WEE1 kinase (or functional homologues such as Mik1) preferentially phosphorylate tyrosine 15 (or 14) on the central cell cycle regulatory protein p34cdc2. Such phosphorylation prevents p34cdc2/cyclin-B complex binding with ATP, effectively blocking the transition from G2 into mitosis. Most of the variations in amino acid sequences of WEE1 are in the amino-terminus, while the carboxy end of these genes are relatively conserved. (Mueller et al. 1995). The carboxyl terminus and the central portion of the WEE1 protein from S. pombe contain the protein kinase domains and sequences crucial for substrate recognition and catalysis (Aligue et al., 1997). The wee1 gene was first isolated in yeast,(Russel and Nurse, 1987) and later in multicellular eukaryotic systems such as humans (Igarashi et al., 1993), Drosophila (Campbell et al., 1995), Xenopus (Mueller et a., 1954) and mouse (Honda et al., 1995). No wee1 homologs have been reported in plants to date.
The invention provides isolated nucleic acids and their encoded proteins that are involved in cell cycle regulation. The invention further provides recombinant expression cassettes, host cells, transgenic plants, and antibody compositions. The present invention provides methods and compositions relating to altering cell cycle protein content, cell cycle progression and/or composition of plants.
Definitions
The term xe2x80x9cisolatedxe2x80x9d refers to material, such as a nucleic acid or a protein, which is: (1) substantially or essentially free from components which normally accompany or interact with the material as found in its naturally occurring environment or (2) if the material is in its natural environment, the material has been altered by deliberate human intervention to a composition and/or placed at a locus in the cell other than the locus native to the material.
As used herein, xe2x80x9cplantxe2x80x9d includes but is not limited to plant cells, plant tissue and plant seeds.
As used herein, xe2x80x9cnucleic acidxe2x80x9d means a polynucleotide and includes single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases. Nucleic acids may also include modified nucleotides that permit correct read through by a polymerase and do not alter the expression of a polypeptide encoded by the polynucleotide.
As used herein, xe2x80x9cpolypeptidexe2x80x9d means proteins, protein fragments, modified proteins, amino acid sequences and synthetic amino acid sequences. The polypeptide can be glycosylated or not.
As used herein, xe2x80x9cpromoterxe2x80x9d includes reference to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription.
By xe2x80x9cfragmentxe2x80x9d is intended a portion of the nucleotide sequence or a portion of the amino acid sequence and hence protein encoded thereby. Preferably fragments of a nucleotide sequence may encode protein fragments that retain the biological activity of the native nucleic acid. However, fragments of a nucleotide sequence which are useful as hybridization probes generally do not encode fragment proteins retaining biological activity. Fragments of a nucleotide sequence are generally greater than 10 nucleotides, preferably at least 20 nucleotides and up to the entire nucleotide sequence encoding the proteins of the invention. Generally probes are less than 1000 nucleotides and preferably less than 500 nucleotides. Fragments of the invention include antisense sequences used to decrease expression of the inventive nucleic acids. Such antisense fragments may vary in length ranging from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, up to and including the entire coding sequence.
By xe2x80x9cvariantsxe2x80x9d is intended substantially similar sequences. Generally, nucleic acid sequence variants of the invention will have at least 50%, 60, 70%, or preferably 80%, more preferably at least 90% and most preferably at least 95% sequence identity to the native nucleotide sequence.
Generally, polypeptide sequence variants of the invention will have at least about 55%, 60%, 70%, 80%, or preferably at least about 90% and more preferably at least about 95% sequence identity to the native protein.
As used herein, xe2x80x9csequence identityxe2x80x9d or xe2x80x9cidentityxe2x80x9d in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide. A polypeptide is substantially identical to a second polypeptide, for example, where the two polypeptides differ only by a conservative substitution.
For purposes of defining the present invention, the Gap 10 program in the Wisconsin Genetics Software Package using default parameters is used, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., USA. The algorithm used for the GAP program is that of Needleman and Wunsch (J. Mol. Biol. 48:443-453 [1970]). The parameters used are as follows: for nucleotide comparisons the gap creation penalty=50, gap extension penalty=3; for amino acid comparisons the gap creation penalty=12, the gap extension penalty=4.
By xe2x80x9cfunctionally equivalentxe2x80x9d is intended that the sequence of the variant defines a chain that produces a protein having substantially the same biological effect as the native protein of interest.